Temporally controlled gene deletion provides a powerful technique for examination of gene function in vivo. To permit use of this technology in the study of cardiac pacemaking, we attempted to generate a mouse line expressing an inducible Cre recombinase selectively in cardiac pacemaker cells. The tamoxifen-inducible CreER(T2) construct was 'knocked in' into the pacemaker channel HCN4 locus. In the absence of inducing agent, recombination was undetectable in HCN4-KiT mice. After injection of tamoxifen, highly selective and efficient recombination was observed in the sinoatrial and atrioventricular node. Expression of Cre and tamoxifen per se did not affect cardiac rhythm, basal heart rate and heart rate modulation. By crossing these animals with floxed HCN4 mice, complete deletion of this gene in the sinoatrial node could be achieved. HCN4-KiT mice represent the first tool for the temporally controlled inactivation of floxed target genes selectively in the conduction system of the murine heart.